Natural diversity screening, assay development, and characterization of nylon-6 enzymatic depolymerization

Successes in biocatalytic polyester recycling have raised the possibility of deconstructing alternative polymers enzymatically, with polyamide (PA) being a logical target due to the array of amide-cleaving enzymes present in nature. Here, we screen 40 potential natural and engineered nylon-hydrolyzing enzymes (nylonases), using mass spectrometry to quantify eight compounds resulting from enzymatic nylon-6 (PA6) hydrolysis. Comparative time-course reactions incubated at 40-70 °C showcase enzyme-dependent variations in product distributions and extent of PA6 film depolymerization, with significant nylon deconstruction activity appearing rare. The most active nylonase, a NylCK variant we rationally thermostabilized (an N-terminal nucleophile (Ntn) hydrolase, NylCK-TS, Tm = 87.4 °C, 16.4 °C higher than the wild-type), hydrolyzes 0.67 wt% of a PA6 film. Reactions fail to restart after fresh enzyme addition, indicating that substrate-based limitations, such as restricted enzyme access to hydrolysable bonds, prohibit more extensive deconstruction. Overall, this study expands our understanding of nylonase activity distribution, indicates that Ntn hydrolases may have the greatest potential for further development, and identifies key targets for progressing PA6 enzymatic depolymerization, including improving enzyme activity, product selectivity, and enhancing polymer accessibility.

Supplementary Table 1.Materials characterization of standard Goodfellow PA6 film (0.2 mm thickness) before and after enzymatic PA6 film deconstruction reactions.The Goodfellow PA6 film (0.2 mm thickness) analyzed here was used in all reactions unless otherwise specified.Summary of DSC, GPC and TGA data collected throughout the study.The molar mass dispersity index is represented by Đ.For the sample preparation, "unmodified" samples were analyzed as received from the supplier or as originally synthesized, apart from DSC measurements, where the PA6 was dried at 40 °C in a vacuum oven for 24 h prior to analysis to remove any residual water, "washed" samples were analyzed following the washing protocol described in the Methods prior to reaction, "no enzyme" samples were incubated in reaction buffer (100 mM NaPi buffer, 150 mM NaCl, pH 7.5) for the described amount of time at the stated temperature in the absence of enzyme, and "+NylCK-TS" samples were analyzed following enzymatic reactions for the described amount of time at the stated temperature (1 µM of enzyme, 0.08 mM enzyme/g PA6 film, 0.65% wt PA6 substrate loading, 100 mM pH 7.5 NaPi buffer with 150 mM NaCl).Dashes represent where no data was collected for that condition set.The mass loss measured during TGA analysis can be attributed to water loss as it occurred at around 100 °C.*All Goodfellow PA6 film used in these analyses came from one batch, as there were small differences between samples of material received from Goodfellow (Supplementary Table 4).Values represent a single measurement (n=1).Supplementary Figure 4. Reaction profile of enzymatic PA6 deconstruction.Total released linear 6-AHA oligomers, represented as 6-AHA monomer equivalents, in reactions with 2 µM NylC K -TS and 13 mg PA6 (0.15 mM enzyme/g PA6 film, 0.65 wt% substrate loading) at 60 °C over the course of 14 days, in reaction buffer (100 mM NaPi buffer, pH 7.5, 150 mM NaCl).Reactions were carried out in triplicate (n=3); error bars represent the standard deviation of the replicate measurements; the error bar centers are the means of the replicate measurements; circles represent the mean value of the triplicate measurements.

Supplementary Figure 5. Release of PA6 oligomers during reaction. A.
There was minimal release of linear oligomers of 6-AHA following incubation of PA6 film (13 mg, 0.65 wt% substrate loading) in reaction buffer (100 mM NaPi, pH 7.5, 150 mM NaCl) over the course of 10 days from 40-70 °C.B. Total released cyclic 6-AHA oligomers following reaction with enzyme (+ Enzyme, 2 µM LCC-ICCG) and 13 mg PA6 (0.15 mM enzyme/g PA6 film, 0.65 wt% substrate loading) or without enzyme (no enzyme) over the course of 10 days (60 °C, 100 mM NaPi buffer, pH 7.5, 150 mM NaCl).Cyclic-oligomer concentrations shown here are representative of all enzymes apart from NylC-type, GatA and UMG-SP-1.For both charts, reactions were carried out in triplicate (n=3), error bars show the standard deviation of the replicate measurements, the error bar centers are the means of the replicate measurements, and the replicate measurements are represented as grey circles.Supplementary Table 2. Stability of linear PA6 oligomer standards following incubation.Chemical standards of 6-AHA monomer, 6-AHA dimer and 6-AHA trimer (10 µg mL -1 ) were individually incubated over the course of 10 days from 40-80 °C in reaction buffer (100 mM NaPi buffer, pH 7.5, 150 mM NaCl).The values represent the percentage recovery of a 10 µg mL -1 standard for each compound following incubation at the stated temperature and duration, as measured by LC-MS/MS.Reactions were conducted in triplicate (n=3), with the S.D. representing the standard deviation of the replicate measurements.

Supplementary Figure 7. Reaction profile of enzymatic PA6 deconstruction with crude cell lysate or purified protein.
Total released linear 6-AHA oligomers, represented as 6-AHA monomer equivalents, in reactions with with either 100 µL of NylC K -TS containing cell lysate (Crude lysate) as the biocatalytic agent, or 1 µM purified NylC K -TS (Purified protein) with 13 mg PA6 (0.08 mM enzyme/g PA6 film in the purified enzyme reaction, 0.65 wt% substrate loading) at 60 °C over the course of 10 days, in reaction buffer (100 mM NaPi buffer, pH 7.5, 150 mM NaCl).Reactions were carried out in triplicate (n=3); error bars represent the standard deviation of the replicate measurements; the error bar centers are the means of the replicate measurements; the replicate measurements are represented as grey circles.
Supplementary Figure 6.SDS-PAGE gel of purified NylC K -TS and NylC K -TS crude cell lysate.NylC K -TS was prepared as either a purified protein or crude cell lysate as detailed in the Methods.Lanes contain the following samples (1) ladder, (2) 1 mg mL -1 NylC K -TS, (3) 0.5 mg mL -1 NylC K -TS, (4) 0.1 mg mL -1 NylCK-TS, (5) 100% crude cell lysate, (6) 50% crude cell lysate, (7) 10% crude cell lysate.Purified NylC K -TS appears as two bands on the gel (~9 kDa and ~27 kDA), as its component subunits dissociate.The major contaminant band in the crude cell lysate samples is assumed to be lysozyme (~15 kDA) from the chemical lysis preparation.The gel was prepared twice, with the gel shown being representative of the set.The uncropped gel is available in Supplementary Figure 32.Supplementary Figure 8.Reactions of NylC-type enzymes with PA6.Total released linear 6-AHA oligomers from enzymes in the NylC-type group, following reaction with PA6 film.Reactions contained 2 µM enzyme and 13 mg PA6 (0.15 mM enzyme/g PA6 film, 0.65 wt% substrate loading) and were incubated from 40-70 °C over the course of 10 days in reaction buffer (100 mM NaPi buffer, pH 7.5, 150 mM NaCl).Above 50 °C, reactions with enzymes that led to no detectable product release above background are not shown.Reactions were carried out in triplicate (n=3), error bars show the standard deviation of the replicate measurements, the error bar centers are the means of the replicate measurements, and the replicate measurements are represented as grey circles.Supplementary Figure 11.Reactions of amidases with PA6.Total released linear 6-AHA oligomers with enzymes in the amidase group following reaction with PA6 film.Reactions contained 2 µM enzyme and 13 mg PA6 (0.15 mM enzyme/g PA6 film, 0.65 wt% substrate loading) and were incubated from 40-70 °C over the course of 10 days in reaction buffer (100 mM NaPi buffer, pH 7.5, 150 mM NaCl).Above 50 °C, reactions with enzymes that led to no detectable product release above background are not shown.Reactions were carried out in triplicate (n=3), error bars show the standard deviation of the replicate measurements, the error bar centers are the means of the replicate measurements, and the replicate measurements are represented as grey circles.Concentrations of 6-AHA cyclic-trimer following reactions of NylC-type enzymes with PA6 film versus a no enzyme control.For enzymatic reactions, reactions contained 2 µM enzyme and 13 mg PA6 (0.15 mM enzyme/g PA6 film, 0.65 wt% substrate loading) and were incubated at the enzyme's optimal temperature (x-axis) over the course of 10 days in reaction buffer (100 mM NaPi buffer, pH 7.5, 150 mM NaCl).No enzyme reactions were treated in the same way, but without enzyme addition.Reactions were carried out in triplicate (n=3), error bars show the standard deviation of the replicate measurements, the error bar centers are the means of the replicate measurements, and the replicate measurements are represented as grey circles.NylB'-SCY   and C. TGA plots for reactions with or without enzyme after 10 days of incubation at 60 °C versus the washed PA6 film prior to reaction.No enzyme reactions contained 13 mg PA6 (0.65 wt% substrate loading) in reaction buffer (100 mM NaPi buffer, pH 7.5, 150 mM NaCl).NylC K -TS reactions contained 1µM NylC K -TS and 13 mg PA6 (0.08 mM enzyme/g PA6 film, 0.65 wt% substrate loading) in reaction buffer (100 mM NaPi buffer, pH 7.5, 150 mM NaCl).For the TGA plots, the darker colored lines represent the weight (%) (left y-axis), and the lighter lines the derivative weight (%) (right y-axis).Plots represent single measurements (n=1).

Supplementary Figure 1 . 2 .
Melt curves for wild-type and thermostabilized NylC variants.Melt curve readings were carried out in triplicate (n=3) by differential scanning fluorimetry (DSF).Dashed lines represent wild-type proteins, solid lines represent thermostabilized proteins.Activity assays with N-(4-nitrophenyl)butanamide (N4NB) for all enzymes.p-NA (pnitroaniline) release profiles of all enzymes tested.Reactions contained 0.5 mM N4NB substrate and 2 µM enzyme in 100 mM sodium phosphate buffer (NaPi), pH 7.5, 150 mM NaCl, and were monitored over the course of 24 h at 30 °C.Enzymes are plotted by grouping: A. NylB-type, B & C. NylC-type, D, E & F. Amidases and Misc., G. Proteases, and H & I. SHD-hydrolases.Points represent the mean of triplicate measurements (n=3), with the error bars representing the standard deviations of replicate measurements being too small to be visualized.Note that y-axes are shown on different scales to enable different levels of activity to be seen.A no enzyme control reaction (No E) is shown in panel A.
Supplementary Figure13.Reactions of AfNitB and RoL10X with PA6.Total released linear 6-AHA oligomers with enzymes not in one of the five main groups (misc.),following reaction with PA6 film.Reactions contained 2 µM enzyme and 13 mg PA6 (0.15 mM enzyme/g PA6 film, 0.65 wt% substrate loading) and were incubated from 40-70 °C over the course of 10 days in reaction buffer (100 mM NaPi buffer, pH 7.5, 150 mM NaCl).Minimal additional product release above background levels was seen under any reaction condition, with representative reactions at 40-50 °C shown.Reactions were carried out in triplicate (n=3), error bars show the standard deviation of the replicate measurements, the error bar centers are the means of the replicate measurements, and the replicate measurements are represented as grey circles.Supplementary Figure12.Reactions of proteases with PA6.Total released linear 6-AHA oligomers with enzymes in the protease group, following reaction with PA6 film.Reactions contained 2 µM enzyme and 13 mg PA6 (0.15 mM enzyme/g PA6 film, 0.65 wt% substrate loading) and were incubated from 40-70 °C over the course of 10 days in reaction buffer (100 mM NaPi buffer, pH 7.5, 150 mM NaCl).Minimal product release above background levels was seen at any reaction condition, with representative reactions at 40-50 °C shown.Reactions were carried out in triplicate (n=3), error bars show the standard deviation of the replicate measurements, the error bar centers are the means of the replicate measurements, and the replicate measurements are represented as grey circles. of NylC K -TS and Tt-NylC with 6-AHA linear oligomers.A. Reaction of NylC K -TS with 6-AHA linear trimer for 24 h at reaction conditions (50 µM 6-AHA trimer, pH 7.5 NaPi buffer, 150 mM NaCl, 2 µM enzyme, 60 °C), showing the change in 6-AHA oligomers as a percentage of total oligomers in the reaction.B. Reaction of Tt-NylC with 6-AHA linear trimer for 24 h at reaction conditions (50 µM 6-AHA trimer, pH 7.5 NaPi buffer, 150 mM NaCl, 2 µM enzyme, 60 °C), showing the change in 6-AHA oligomers as a percentage of total oligomers in the reaction.C. Reaction of NylC K -TS with 6-AHA linear dimer for 24 h at reaction conditions (100 µM 6-AHA dimer, pH 7.5 NaPi buffer, 150 mM NaCl, 2 µM enzyme, 60 °C), showing the change in 6-AHA oligomers as a percentage of total oligomers in the reaction.For both 6-AHA dimer and 6-AHA trimer, there was no change in concentration in no enzyme control reactions.For A, B, and C, reactions were carried out in triplicate (n=3); error bars represent the standard deviation of the replicate measurements, and the error bar centers are the means of the replicate measurements.cyclic-trimer in reactions with the most active NylCs.
Supplementary Figure16.Comparative reactions of NylC K and NylC K -TS.Total released linear 6-AHA oligomers with NylC K and NylC K -TS.Reactions contained 1 µM enzyme and 13 mg PA6 (0.08 mM enzyme/g PA6 film, 0.65 wt% substrate loading) and were incubated from 40-80 °C over the course of 10 days in reaction buffer (100 mM NaPi buffer, pH 7.5, 150 mM NaCl).Graphs to the right present a zoom in of the first seven hours of reaction.Reactions were carried out in triplicate (n=3), error bars show the standard deviation of the replicate measurements, and the error bar centers are the means of the replicate measurements.
Reaction of NylB'-SCY with 6-AHA linear dimer for 24 h at reaction conditions (100 µM 6-AHA dimer, pH 7.5 NaPi buffer, 150 mM NaCl, 2 µM enzyme, 70 °C), showing the change in 6-AHA oligomers as a percentage of total oligomers in the reaction.B. Reaction of NylB'-SCY with 6-AHA linear trimer for 24 h at reaction conditions (50 µM 6-AHA trimer, pH 7.5 NaPi buffer, 150 mM NaCl, 2 µM enzyme, 70 °C), showing the change in 6-AHA oligomers as a percentage of total oligomers in the reaction.C. Reaction of LCC-ICCG with 6-AHA linear trimer for 24 h at reaction conditions (50 µM 6-AHA trimer, pH 7.5 NaPi buffer, 150 mM NaCl, 2 µM enzyme, 70 °C), showing the change in 6-AHA oligomers as a percentage of total oligomers in the reaction.For A, B, and C, reactions were carried out in triplicate (n=3); error bars represent the standard deviation of the replicate measurements; the error bar centers are the means of the replicate measurements.
characterization of PA6 films incubated with and without enzyme.Comparison of A. GPC, measured with the differential refractive index (dRI) detector B. DSC,

. Reactions of NylB-type enzymes with PA6.
Total released linear 6-AHA oligomers from enzymes in the NylB-type group, following reaction with PA6 film.Reactions contained 2 µM enzyme and 13 mg PA6 (0.15 mM enzyme/g PA6 film, 0.65 wt% substrate loading) and were incubated from 40-70 °C over the course of 10 days in reaction buffer (100 mM NaPi buffer, pH 7.5, 150 mM NaCl).Above 50 °C, minimal product release above background was seen with any of the NylB-type enzymes.Reactions were carried out in triplicate (n=3), error bars show the standard deviation of the replicate measurements, the error bar centers are the means of the replicate measurements, and the replicate measurements are represented as grey circles.
Supplementary Figure10.Reactions of SHD-hydrolases with PA6.Total released linear 6-AHA oligomers with enzymes in the SHD-hydrolase group following reaction with PA6 film.Reactions contained 2 µM enzyme and 13 mg PA6 (0.15 mM enzyme/g PA6 film, 0.65 wt% substrate loading) and were incubated from 40-70 °C over the course of 10 days in reaction buffer (100 mM NaPi buffer, pH 7.5, 150 mM NaCl).Above 50 °C, reactions with enzymes that led to no detectable product release above background are not shown.Reactions were carried out in triplicate (n=3), error bars show the standard deviation of the replicate measurements, the error bar centers are the means of the replicate measurements, and the replicate measurements are represented as grey circles.

Supplementary Figure 23. Crystallinity changes in no enzyme control reactions over time at different temperatures.
Crystallinity changes of PA6 film were determined by DSC over the course of 10 days.For each temperature from 40-70 °C, 13 mg PA6 film (0.65 wt% substrate loading) was incubated in reaction buffer (100 mM NaPi buffer, pH 7.5, 150 mM NaCl).Points represent single measurements (n=1).
error bars represent the standard deviation of the replicate measurements; circle, square, and triangle points represent the mean value of the triplicate measurements; the error bar centers are the means of the replicate measurements.K -TS and 13 mg PA6 (0.08 mM enzyme/g PA6 film, 0.65 wt% substrate loading, normal reaction), where following reaction for 7 days, either fresh enzyme (1µM) or fresh PA6 film (13 mg) was added, and the reaction allowed to progress.For A, B, C, and D reactions were conducted in 100 mM NaPi buffer, pH 7.5, 150 mM NaCl at 60 °C; reactions were carried out in triplicate (n=3); error bars represent the standard deviation of the replicate measurements; circle, square and triangle points represent the mean value of the triplicate measurements; the error bar centers are the means of the replicate measurements.